Melioidosis caused by the gram-negative bacterium Burkholderia pseudomallei is remarkably resistant to most classes of antibiotics. Even after months of treatment with antibacterials relatively effective in vitro, between 6 and 20% of individuals suffer from relapsed B. pseudomallei infections, indicating that this pathogen alters its patterns of antibacterial susceptibility in response to cues encountered in the host. Data presented in our application have identified nitrosative stress and anaerobiosis as two host conditions that promote B. pseudomallei drug tolerance. Our goal is to identify antibacterials that improve the effectiveness of therapy against melioidosis and identify novel drug targets. We hypothesize that nitric oxide and low oxygen tensions drive B. pseudomallei into an altered metabolic and drug-tolerant state. To address this hypothesis we will determine the: 1) molecular adaptations that mediate resistance of B. pseudomallei to nitrosative stress; 2) adaptive mechanisms that facilitate anaerobic survival of B. pseudomallei; and 3) contribution of nitrosative stress and anaerobic stasis to antibacterial tolerance. Our recently developed bacteriological techniques, genetics, microarray transcriptional profiling, cell culture systems and biochemical assays will help us identify adaptive and metabolic pathways that promote tolerance of 8. pseudomallei to antibacterials used in humans suffering from melioidosis. In collaboration with Dr. Steven Dow at Colorado State University, we will determine how antinitrosative defenses and metabolic networks contribute to the pathogenesis of 8. pseudomallei in a murine model of melioidosis. In addition, we will continue with our efforts to identify antibacterials effective against nonrespiring 8. pseudomallei. Towards this goal, we have already determined that drugs that target anaerobes are very effective against nonrespiring 8. pseudomallei while drugs that kill aerobic bacteria fail. The therapeutic potential of anaerobic antibacterials against nonrespiring 8. pseudomallei will be studied in a murine model of chronic melioidosis. These studies are within the scope of the RMRCE to discover novel antibacterial targets and to develop antibacterials that improve efficacy against the widespread clinical antibiotic resistance of melioidosis. Our investigations synergize with the research proposed by Dr. Schweizer in project RP 2.1 to study the natural resistance of 8. pseudomallei to antibacterials tested under aerobic conditions in a murine model of acute infection